The present invention relates to the field of molecular biology. In particular, it relates to, among other things, nucleotide sequences of Staphylococcus aureus, contigs, ORFs, fragments, probes, primers and related polynucleotides thereof, peptides and polypeptides encoded by the sequences, and uses of the polynucleotides and sequences thereof, such as in fermentation, polypeptide production, assays and pharmaceutical development, among others.
The genus Staphylococcus includes at least 20 distinct species. (For a review see Novick, R. P., The Staphylococcus as a Molecular Genetic System, Chapter 1, pgs. 1-37 in MOLECULAR BIOLOGY OF THE STAPHYLOCOCCI, R. Novick, Ed., VCH Publishers, New York (1990)). Species differ from one another by 80% or more, by hybridization kinetics, whereas strains within a species are at least 90% identical by the same measure.
The species Staphylococcus aureus, a gram-positive, facultatively aerobic, clump-forming cocci, is among the most important etiological agents of bacterial infection in humans, as discussed briefly below.
Human Health and S. Aureus 
Staphylococcus aureus is a ubiquitous pathogen. (See, for instance, Mims et al., MEDICAL MICROBIOLOGY, Mosby-Year Book Europe Limited, London, UK (1993)). It is an etiological agent of a variety of conditions, ranging in severity from mild to fatal. A few of the more common conditions caused by S. aureus infection are burns, cellulitis, eyelid infections, food poisoning, joint infections, neonatal conjunctivitis, osteomyelitis, skin infections, surgical wound infection, scalded skin syndrome and toxic shock syndrome, some of which are described further below.
Burns
Burn wounds generally are sterile initially. However, they generally compromise physical and immune barriers to infection, cause loss of fluid and electrolytes and result in local or general physiological dysfunction. After cooling, contact with viable bacteria results in mixed colonization at the injury site. Infection may be restricted to the non-viable debris on the burn surface (xe2x80x9cescharxe2x80x9d), it may progress into full skin infection and invade viable tissue below the eschar and it may reach below the skin, enter the lymphatic and blood circulation and develop into septicaemia. S. aureus is among the most important pathogens typically found in burn wound infections. It can destroy granulation tissue and produce severe septicaemia.
Cellulitis
Cellulitis, an acute infection of the skin that expands from a typically superficial origin to spread below the cutaneous layer, most commonly is caused by S. aureus in conjunction with S. pyrogenes. Cellulitis can lead to systemic infection. In fact, cellulitis can be one aspect of synergistic bacterial gangrene. This condition typically is caused by a mixture of S. aureus and microaerophilic streptococci. It causes necrosis and treatment is limited to excision of the necrotic tissue. The condition often is fatal.
Eyelid Infections
S. aureus is the cause of styes and of sticky eyexe2x80x9d in neonates, among other eye infections. Typically such infections are limited to the surface of the eye, and may occasionally penetrate the surface with more severe consequences.
Food Poisoning
Some strains of S. aureus produce one or more of five serologically distinct, heat and acid stable enterotoxins that are not destroyed by digestive process of the stomach and small intestine (enterotoxins A-E). Ingestion of the toxin, in sufficient quantities, typically results in severe vomiting, but not diarrhoea. The effect does not require viable bacteria. Although the toxins are known, their mechanism of action is not understood.
Joint Infections
S. aureus infects bone joints causing diseases such osteomyelitis.
Osteomyelitis
S. aureus is the most common causative agent of haematogenous osteomyelitis. The disease tends to occur in children and adolescents more than adults and it is associated with non-penetrating injuries to bones. Infection typically occurs in the long end of growing bone, hence its occurrence in physically immature populations. Most often, infection is localized in the vicinity of sprouting capillary loops adjacent to epiphysial growth plates in the end of long, growing bones.
Skin Infections
S. aureus is the most common pathogen of such minor skin infections as abscesses and boils. Such infections often are resolved by normal host response mechanisms, but they also can develop into severe internal infections. Recurrent infections of the nasal passages plague nasal carriers of S. aureus. 
Surgical Wound Infections
Surgical wounds often penetrate far into the body. Infection of such wound thus poses a grave risk to the patient. S. aureus is the most important causative agent of infections in surgical wounds. S. aureus is unusually adept at invading surgical wounds; sutured wounds can be infected by far fewer S. aureus cells then are necessary to cause infection in normal skin. Invasion of surgical wound can lead to severe S. aureus septicaemia. Invasion of the blood stream by S. aureus can lead to seeding and infection of internal organs, particularly heart valves and bone, causing systemic diseases, such as endocarditis and osteomyelitis.
Scalded Skin Syndrome
S. aureus is responsible for xe2x80x9cscalded skin syndromexe2x80x9d (also called toxic epidermal necrosis, Ritter""s disease and Lyell""s disease). This diseases occurs in older children, typically in outbreaks caused by flowering of S. aureus strains produce exfoliation (also called scalded skin syndrome toxin). Although the bacteria initially may infect only a minor lesion, the toxin destroys intercellular connections, spreads epidermal layers and allows the infection to penetrate the outer layer of the skin, producing the desquamation that typifies the diseases. Shedding of the outer layer of skin generally reveals normal skin below, but fluid lost in the process can produce severe injury in young children if it is not treated properly.
Toxic Shock Syndrome
Toxic shock syndrome is caused by strains of S. aureus that produce the so-called toxic shock syndrome toxin. The disease can be caused by S. aureus infection at any site, but it is too often erroneously viewed exclusively as a disease solely of women who use tampons. The disease involves toxaemia and septicaemia, and can be fatal.
Nocosomial Infections
In the 1984 National Nocosomial Infection Surveillance Study (xe2x80x9cNNISxe2x80x9d) S. aureus was the most prevalent agent of surgical wound infections in many hospital services, including medicine, surgery, obstetrics, pediatrics and newborns.
Resistance to Drugs of S. aureus Strains
Prior to the introduction of penicillin the prognosis for patients seriously infected with S. aureus was unfavorable. Following the introduction of penicillin in the early 1940s even the worst S. aureus infections generally could be treated successfully. The emergence of penicillin-resistant strains of S. aureus did not take long, however. Most strains of S. aureus encountered in hospital infections today do not respond to penicillin; although, fortunately, this is not the case for S. aureus encountered in community infections.
It is well known now that penicillin-resistant strains of S. aureus produce a lactamase which converts penicillin to pencillinoic acid, and thereby destroys antibiotic activity. Furthermore, the lactamase gene often is propagated episomally, typically on a plasmid, and often is only one of several genes on an episomal element that, together, confer multidrug resistance.
Methicillins, introduced in the 1960s, largely overcame the problem of penicillin resistance in S. aureus. These compounds conserve the portions of penicillin responsible for antibiotic activity and modify or alter other portions that make penicillin a good substrate for inactivating lactamases. However, methicillin resistance has emerged in S. aureus, along with resistance to many other antibiotics effective against this organism, including aminoglycosides, tetracycline, chloramphenicol, macrolides and lincosamides. In fact, methicillin-resistant strains of S. aureus generally are multiply drug resistant.
The molecular genetics of most types of drug resistance in S. aureus has been elucidated (See Lyon et al., Microbiology Reviews 51: 88-134 (1987)). Generally, resistance is mediated by plasmids, as noted above regarding penicillin resistance; however, several stable forms of drug resistance have been observed that apparently involve integration of a resistance element into the S. aureus genome itself.
Thus far each new antibiotic gives rise to resistance strains, stains emerge that are resistance to multiple drugs and increasingly persistent forms of resistance begin to emerge. Drug resistance of S. aureus infections already poses significant treatment difficulties, which are likely to get much worse unless new therapeutic agents are developed.
Molecular Genetics of Staphylococcus Aureus 
Despite its importance in, among other things, human disease, relatively little is known about the genome of this organism.
Most genetic studies of S. aureus have been carried out using the the strain NCTC8325, which contains prophages psil1, psil2 and psil3, and the UV-cured derivative of this strain, 8325-4 (also referred to as RN450), which is free of the prophages.
These studies revealed that the S. aureus genome, like that of other staphylococci, consists of one circular, covalently closed, double-stranded DNA and a collection of so-called variable accessory genetic elements, such as prophages, plasmids, transposons and the like.
Physical characterization of the genome has not been carried out in any detail. Pattee et al. published a low resolution and incomplete genetic and physical map of the chromosome of S. aureus strain NCTC 8325. (Pattee et al. Genetic and Physical Mapping of Chromosome of Staphylococcus aureus NCTC 8325, Chapter 11, pgs. 163-169 in.MOLECULAR BIOLOGY OF THE STAPHYLOCOCCI, R. P. Novick, Ed., VCH Publishers, New York, (1990) The genetic map largely was produced by mapping insertions of Tn551 and Tn4001, which, respectively, confer erythromycin and gentamicin resistance, and by analysis of SmaI-digested DNA by Pulsed Field Gel Electrophoresis (xe2x80x9cPFGExe2x80x9d).
The map was of low resolution; even estimating the physical size of the genome was difficult, according to the investigators. The size of the largest SmaI chromosome fragment, for instance, was too large for accurate sizing by PFGE. To estimate its size, additional restriction sites had to be introduced into the chromosome using a transposon containing a SmaI recognition sequence.
In sum, most physical characteristics and almost all of the genes of Staphylococcus aureus are unknown. Among the few genes that have been identified, most have not been physically mapped or characterized in detail. Only a very few genes of this organism have been sequenced. (See, for instance Thornsberry, J., Antimicrobial Chemotherapy 21 Suppl C: 9-16 (1988), current versions of GENBANK and other nucleic acid databases, and references that relate to the genome of S. aureus such as those set out elsewhere herein.)
It is clear that the etiology of diseases mediated or exacerbated by S. aureus infection involves the programmed expression of S. aureus genes, and that characterizing the genes and their patterns of expression would add dramatically to our understanding of the organism and its host interactions. Knowledge of S. aureus genes and genomic organization would dramatically improve understanding of disease etiology and lead to improved and new ways of preventing, ameliorating, arresting and reversing diseases. Moreover, characterized genes and genomic fragments of S. aureus would provide reagents for, among other things, detecting, characterizing and controlling S. aureus infections. There is a need therefore to characterize the genome of S. aureus and for polynucleotides and sequences of this organism.
The present invention is based on the sequencing of fragments of the Staphylococcus aureus genome. The primary nucleotide sequences which were generated are provided in SEQ ID NOS:1-5,191.
The present invention provides the nucleotide sequence of several thousand contigs of the Staphylococcus aureus genome, which are listed in tables below and set out in the Sequence Listing submitted herewith, and representative fragments thereof, in a form which can be readily used, analyzed, and interpreted by a skilled artisan. In one embodiment, the present invention is provided as contiguous strings of primary sequence information corresponding to the nucleotide sequences depicted in SEQ ID NOS:1-5,191.
The present invention further provides nucleotide sequences which are at least 95% identical to the nucleotide sequences of SEQ ID NOS:1-5,191.
The nucleotide sequence of SEQ ID NOS:1-5,191, a representative fragment thereof, or a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NOS:1-5,191 may be provided in a variety of mediums to facilitate its use. In one application of this embodiment, the sequences of the present invention are recorded on computer readable media. Such media includes, but is not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
The present invention further provides systems, particularly computer-based systems which contain the sequence information herein described stored in a data storage means. Such systems are designed to identify commercially important fragments of the Staphylococcus aureus genome.
Another embodiment of the present invention is directed to fragments of the Staphylococcus aureus genome having particular structural or functional attributes. Such fragments of the Staphylococcus aureus genome of the present invention include, but are not limited to, fragments which encode peptides, hereinafter referred to as open reading frames or ORFs,xe2x80x9d fragments which modulate the expression of an operably linked ORF, hereinafter referred to as expression modulating fragments or EMFs,xe2x80x9d and fragments which can be used to diagnose the presence of Staphylococcus aureus in a sample, hereinafter referred to as diagnostic fragments or xe2x80x9cDFs.xe2x80x9d
Each of the ORFs in fragments of the Staphylococcus aureus genome disclosed in Tables 1-3, and the EMFs found 5xe2x80x2 to the ORFs, can be used in numerous ways as polynucleotide reagents. For instance, the sequences can be used as diagnostic probes or amplification primers for detecting or determining the presence of a specific microbe in a sample, to selectively control gene expression in a host and in the production of polypeptides, such as polypeptides encoded by ORFs of the present invention, particular those polypeptides that have a pharmacological activity.
The present invention further includes recombinant constructs comprising one or more fragments of the Staphylococcus aureus genome of the present invention. The recombinant constructs of the present invention comprise vectors, such as a plasmid or viral vector, into which a fragment of the Staphylococcus aureus has been inserted.
The present invention further provides host cells containing any of the isolated fragments of the Staphylococcus aureus genome of the present invention. The host cells can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic cell, such as a yeast cell, or a procaryotic cell such as a bacterial cell.
The present invention is further directed to isolated polypeptides and proteins encoded by ORFs of the present invention. A variety of methods, well known to those of skill in the art, routinely may be utilized to obtain any of the polypeptides and proteins of the present invention. For instance, polypeptides and proteins of the present invention having relatively short, simple amino acid sequences readily can be synthesized using commercially available automated peptide synthesizers. Polypeptides and proteins of the present invention also may be purified from bacterial cells which naturally produce the protein. Yet another alternative is to purify polypeptide and proteins of the present invention can from cells which have been altered to express them.
The invention further provides polypeptides comprising Staphylococcus aureus epitopes and vaccine compositions comprising such polypeptides. Also provided are methods for vacciniating an individual against Staphylococcus aureus infection.
The invention further provides methods of obtaining homologs of the fragments of the Staphylococcus aureus genome of the present invention and homologs of the proteins encoded by the ORFs of the present invention. Specifically, by using the nucleotide and amino acid sequences disclosed herein as a probe or as primers, and techniques such as PCR cloning and colony/plaque hybridization, one skilled in the art can obtain homologs.
The invention further provides antibodies which selectively bind polypeptides and proteins of the present invention. Such antibodies include both monoclonal and polyclonal antibodies.
The invention further provides hybridomas which produce the above-described antibodies. A hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.
The present invention further provides methods of identifying test samples derived from cells which express one of the ORFs of the present invention, or a homolog thereof. Such methods comprise incubating a test sample with one or more of the antibodies of the present invention, or one or more of the Dfs or antigens of the present invention, under conditions which allow a skilled artisan to determine if the sample contains the ORF or product produced therefrom.
In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the above-described assays.
Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the antibodies, antigens, or one of the DFs of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of bound antibodies, antigens or hybridized DFs.
Using the isolated proteins of the present invention, the present invention further provides methods of obtaining and identifying agents capable of binding to a polypeptide or protein encoded by one of the ORFs of the present invention. Specifically, such agents include, as further described below, antibodies, peptides, carbohydrates, pharmaceutical agents and the like. Such methods comprise steps of: (a) contacting an agent with an isolated protein encoded by one of the ORFs of the present invention; and (b) determining whether the agent binds to said protein.
The present genomic sequences of Staphylococcus aureus will be of great value to all laboratories working with this organism and for a variety of commercial purposes. Many fragments of the Staphylococcus aureus genome will be immediately identified by similarity searches against GenBank or protein databases and will be of immediate value to Staphylococcus aureus researchers and for immediate commercial value for the production of proteins or to control gene expression.
The methodology and technology for elucidating extensive genomic sequences of bacterial and other genomes has and will greatly enhance the ability to analyze and understand chromosomal organization. In particular, sequenced contigs and genomes will provide the models for developing tools for the analysis of chromosome structure and function, including the ability to identify genes within large segments of genomic DNA, the structure, position, and spacing of regulatory elements, the identification of genes with potential industrial applications, and the ability to do comparative genomic and molecular phylogeny.